Genetic regulation of surfactant deficiency has been suggested by heritability of neonatal respiratory distress syndrome, persistence of gender and racial disparities in disease risk, and the frequency of surfactant replacement treatment failures. Individually rare, collectively common (2-4/100 infants) mutations in the ATP- binding cassette sub-family A member 3 gene (ABCA3) disrupt surfactant function through diverse mechanisms including reduced lipid transport, ABCA3 misfolding or altered trafficking, or induction of increased endoplasmic reticulum stress and increase risk for neonatal respiratory distress syndrome in term and late preterm infants. Our goal is to develop and implement a human model system that uses static and dynamic lipidomic signatures for functional screening of ABCA3 mutations and that could be used for testing small molecules to correct mutation-encoded, functional defects in any gene expressed in the human alveolar type 2 cell. In the R21 Phase of this Award, using adenoviral vectors with high transduction efficiency and cargo capacity and highly sensitive and specific mass spectrometry based lipidomic profiling, we will test the hypothesis that ABCA3 gene silencing and rescue with wild-type ABCA3 cDNA reconstitute surfactant phospholipid signatures in human, primary alveolar type 2 cells. In the R33 Phase of this Award, using mass spectrometry-based lipidomic profiling, electron microscopy, and surface activity measurements, we will examine disruption of surfactant lipidomic signatures, lipid turnover rates, and lipid secretion kinetics, lamellar body phenotype, and surfactant function by previously characterized and uncharacterized ABCA3 mutations associated with increased risk for neonatal respiratory distress syndrome. The overall impact of this Award will provide a human model system for functional, lipidomics-based screening of genomic hits associated with surfactant deficiency and for development of small- molecule based strategies to correct mutation-encoded, functional surfactant defects.